Brain energy metabolism: A roadmap for future research.

Although we have learned much about how the brain fuels its functions over the last decades, there remains much still to discover in an organ that is so complex. This article lays out major gaps in our knowledge of interrelationships between brain metabolism and brain function, including biochemical, cellular, and subcellular aspects of functional metabolism and its imaging in adult brain, as well as during development, aging, and disease. The focus is on unknowns in metabolism of major brain substrates and associated transporters, the roles of insulin and of lipid droplets, the emerging role of metabolism in microglia, mysteries about the major brain cofactor and signaling molecule NAD+, as well as unsolved problems underlying brain metabolism in pathologies such as traumatic brain injury, epilepsy, and metabolic downregulation during hibernation. It describes our current level of understanding of these facets of brain energy metabolism as well as a roadmap for future research.

Case for supporting astrocyte energetics in glucose transporter 1 deficiency syndrome.

In glucose transporter 1 deficiency syndrome (Glut1DS), glucose transport into brain is reduced due to impaired Glut1 function in endothelial cells at the blood-brain barrier. This can lead to shortages of glucose in brain and is thought to contribute to seizures. Ketogenic diets are the first-line treatment and, among many beneficial effects, provide auxiliary fuel in the form of ketone bodies that are largely metabolized by neurons. However, Glut1 is also the main glucose transporter in astrocytes. Here, we review data indicating that glucose shortage may also impact astrocytes in addition to neurons and discuss the expected negative biochemical consequences of compromised astrocytic glucose transport for neurons. Based on these effects, auxiliary fuels are needed for both cell types and adding medium chain triglycerides (MCTs) to ketogenic diets is a biochemically superior treatment for Glut1DS compared to classical ketogenic diets. MCTs provide medium chain fatty acids (MCFAs), which are largely metabolized by astrocytes and not neurons. MCFAs supply energy and contribute carbons for glutamine and γ-aminobutyric acid synthesis, and decanoic acid can also block α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid glutamate receptors. MCTs do not compete with metabolism of ketone bodies mostly occurring in neurons. Triheptanoin, an anaplerotic but also gluconeogenic uneven MCT, may be another potential addition to ketogenic diets, although maintenance of "ketosis" can be difficult. Gene therapy has also targeted both endothelial cells and astrocytes. Other approaches to increase fuel delivery to the brain currently investigated include exchange of Glut1DS erythrocytes with healthy cells, infusion of lactate, and pharmacological improvement of glucose transport. In conclusion, although it remains difficult to assess impaired astrocytic energy metabolism in vivo, astrocytic energy needs are most likely not met by ketogenic diets in Glut1DS. Thus, we propose prospective studies including monitoring of blood MCFA levels to find optimal doses for add-on MCT to ketogenic diets and assessing of short- and long-term outcomes.

Transient anticonvulsant effects of time-restricted feeding in the 6-Hz mouse model.

INTRODUCTION: Intermittent fasting enhances neural bioenergetics, is neuroprotective, and elicits antioxidant effects in various animal models. There are conflicting findings on seizure protection, where intermittent fasting regimens often cause severe weight loss resembling starvation which is unsustainable long-term. Therefore, we tested whether a less intensive intermittent fasting regimen such as time-restricted feeding (TRF) may confer seizure protection. METHODS: Male CD1 mice were assigned to either ad libitum-fed control, continuous 8 h TRF, or 8 h TRF with weekend ad libitum food access (2:5 TRF) for one month. Body weight, food intake, and blood glucose levels were measured. Seizure thresholds were determined at various time points using 6-Hz and maximal electroshock seizure threshold (MEST) tests. Protein levels and mRNA expression of genes, enzyme activity related to glucose metabolism, as well as mitochondrial dynamics were assessed in the cortex and hippocampus. Markers of antioxidant defence were evaluated in the plasma, cortex, and liver. RESULTS: Body weight gain was similar in the ad libitum-fed and TRF mouse groups. In both TRF regimens, blood glucose levels did not change between the fed and fasted state and were higher during fasting than in the ad libitum-fed groups. Mice in the TRF group had increased seizure thresholds in the 6-Hz test on day 15 and on day 19 in a second cohort of 2:5 TRF mice, but similar seizure thresholds at other time points compared to ad libitum-fed mice. Continuous TRF did not alter MEST seizure thresholds on day 28. Mice in the TRF group showed increased maximal activity of pyruvate dehydrogenase in the cortex, which was accompanied by increased protein levels of mitochondrial pyruvate carrier 1 in the cortex and hippocampus. There were no other major changes in protein or mRNA levels associated with energy metabolism and mitochondrial dynamics in the brain, nor markers of antioxidant defence in the brain, liver, or plasma. CONCLUSIONS: Both continuous and 2:5 TRF regimens transiently increased seizure thresholds in the 6-Hz model at around 2 weeks, which coincided with stability of blood glucose levels during the fed and fasted periods. Our findings suggest that the lack of prolonged anticonvulsant effects in the acute electrical seizure models employed may be attributed to only modest metabolic and antioxidant adaptations found in the brain and liver. Our findings underscore the potential therapeutic value of TRF in managing seizure-related conditions.

Metabolic aspects of genetic ion channel epilepsies.

Nowadays, particularly in countries with high incomes, individual mutations in people affected by genetic epilepsies are identified, and genetic therapies are being developed. In addition, drugs are being screened to directly target specific mutations, and personalised medicine is possible. However, people with epilepsy do not yet benefit from these advances, and many types of epilepsies are medication-resistant, including Dravet syndrome. Thus, in the meantime, alternative and effective treatment options are needed. There is increasing evidence that metabolic deficits contribute to epileptic seizures and that such metabolic impairments may be amenable to treatment, with metabolic treatment options like the ketogenic diet being employed with some success. However, the brain metabolic alterations that occur in ion channel epilepsies are not well-understood, nor how these may differ from epilepsies that are of acquired and unknown origins. Here, we provide an overview of studies investigating metabolic alterations in epilepsies caused by mutations in the SCN1A and KCNA1 genes, which are currently the most studied ion channel epilepsies in animal models. The metabolic changes found in these models are likely to contribute to seizures. A metabolic basis of these ion channel epilepsies is supported by human and/or animal studies that show beneficial effects of the ketogenic diet, which may be mediated by the provision of auxiliary brain fuel in the form of ketone bodies. Other potentially more preferred dietary therapies including medium-chain triglycerides and triheptanoin have also been tested in a limited number of studies, but their efficacies remain to be clearly established. The extent to which brain metabolism is affected in people with Dravet syndrome, KCNA1 epilepsy and the models thereof still requires clarification. This requires more experiments that yield functional insight into metabolism.

Potential new roles for glycogen in epilepsy.

Seizures often originate in epileptogenic foci. Between seizures (interictally), these foci and some of the surrounding tissue often show low signals with 18 fluorodeoxyglucose (FDG) positron emission tomography (PET) in many epileptic patients, even when there are no radiologically detectable structural abnormalities. Low FDG-PET signals are thought to reflect glucose hypometabolism. Here, we review knowledge about metabolism of glucose and glycogen and oxidative stress in people with epilepsy and in acute and chronic rodent seizure models. Interictal brain glucose levels are normal and do not cause apparent glucose hypometabolism, which remains unexplained. During seizures, high amounts of fuel are needed to satisfy increased energy demands. Astrocytes consume glycogen as an additional emergency fuel to supplement glucose during high metabolic demand, such as during brain stimulation, stress, and seizures. In rodents, brain glycogen levels drop during induced seizures and increase to higher levels thereafter. Interictally, in people with epilepsy and in chronic epilepsy models, normal glucose but high glycogen levels have been found in the presumed brain areas involved in seizure generation. We present our new hypothesis that as an adaptive response to repeated episodes of high metabolic demand, high interictal glycogen levels in epileptogenic brain areas are used to support energy metabolism and potentially interictal neuronal activity. Glycogenolysis, which can be triggered by stress or oxidative stress, leads to decreased utilization of plasma glucose in epileptogenic brain areas, resulting in low FDG signals that are related to functional changes underlying seizure onset and propagation. This is (partially) reversible after successful surgery. Last, we propose that potential interictal glycogen depletion in epileptogenic and surrounding areas may cause energy shortages in astrocytes, which may impair potassium buffering and contribute to seizure generation. Based on these hypotheses, auxiliary fuels or treatments that support glycogen metabolism may be useful to treat epilepsy.

ß-Hydroxybutyrate and Medium-Chain Fatty Acids are Metabolized by Different Cell Types in Mouse Cerebral Cortex Slices.

Ketogenic diets and medium-chain triglycerides are gaining attention as treatment of neurological disorders. Their major metabolites, ß-hydroxybutyrate (ßHB) and the medium-chain fatty acids (MCFAs) octanoic acid (C8) and decanoic acid (C10), are auxiliary brain fuels. To which extent these fuels compete for metabolism in different brain cell types is unknown. Here, we used acutely isolated mouse cerebral cortical slices to (1) compare metabolism of 200 µM [U-13C]C8, [U-13C]C10 and [U-13C]ßHB and (2) assess potential competition between metabolism of ßHB and MCFAs by quantifying metabolite 13C enrichment using gas chromatography-mass spectrometry (GC-MS) analysis. The 13C enrichment in most metabolites was similar with [U-13C]C8 and [U-13C]C10 as substrates, but several fold lower with [U-13C]ßHB. The 13C enrichment in glutamate was in a similar range for all three substrates, whereas the 13C enrichments in citrate and glutamine were markedly higher with both [U-13C]C8 and [U-13C]C10 compared with [U-13C]ßHB. As citrate and glutamine are indicators of astrocytic metabolism, the results indicate active MCFA metabolism in astrocytes, while ßHB is metabolized in a different cellular compartment. In competition experiments, 12C-ßHB altered 13C incorporation from [U-13C]C8 and [U-13C]C10 in only a few instances, while 12C-C8 and 12C-C10 only further decreased the low [U-13C]ßHB-derived 13C incorporation into citrate and glutamine, signifying little competition for oxidative metabolism between ßHB and the MCFAs. Overall, the data demonstrate that ßHB and MCFAs are supplementary fuels in different cellular compartments in the brain without notable competition. Thus, the use of medium-chain triglycerides in ketogenic diets is likely to be beneficial in conditions with carbon and energy shortages in both astrocytes and neurons, such as GLUT1 deficiency.

Glyceryl triacetate feeding in mice increases plasma acetate levels but has no anticonvulsant effects in acute electrical seizure models.

INTRODUCTION: Acetate has been shown to have neuroprotective and anti-inflammatory effects. It is oxidized by astrocytes and can thus provide auxiliary energy to the brain in addition to glucose. Therefore, we hypothesized that it may protect against seizures, which is investigated here by feeding glyceryl triacetate (GTA), to provide high amounts of acetate without raising sodium or acid levels. METHOD: CD1 male mice were fed controlled diets with or without GTA for up to three weeks. Body weights, blood glucose levels, plasma short-chain fatty acid levels, and other hematological parameters were monitored. Seizure thresholds were determined in 6 Hz and maximal electroshock seizure threshold (MEST) tests. Antioxidant capacities were evaluated in the cerebral cortex and plasma using a ferric reducing antioxidant power (FRAP) assay and Trolox equivalent antioxidant capacity assay. RESULTS: Body weight gain was similar with both diets with and without GTA in two experiments. Glyceryl triacetate-fed groups showed 2-3- and 1.6-fold increased acetate and propionate levels in plasma, respectively. Glucose levels were unaltered in blood collected from the tail tip but increased in trunk blood. No differences were found in the activity of cerebral cortex acetyl-CoA synthetase. In the 6 Hz threshold test, seizure thresholds were lower by 3 mA and 2.4 mA after 8 and 14 days, respectively, in the GTA compared to the control diet-fed group, but showed no difference on day 16, showing that GTA has small, but inconsistent proconvulsant effects in this model. In MEST tests, a slightly increased seizure threshold (1 mA) was found on day 19 in the GTA-fed group, but not in another experiment on day 21. There were no differences in antioxidant capacity in plasma or cortex between the two groups. CONCLUSION: Glyceryl triacetate feeding showed no antioxidant effects nor beneficial changes in acute electrical seizure threshold mouse models, despite its ability to increase plasma acetate levels.

Dietary medium chain triglycerides for management of epilepsy: New data from human, dog, and rodent studies.

Many studies show that glucose metabolism in epileptic brain areas can be impaired. Energy is crucial to maintain normal brain function, including ion and neurotransmitter balances. Energy deficits can lead to disruption of ion gradients, which can trigger neuronal depolarization and generation of seizures. Thus, perturbed metabolic processing of glucose in epileptogenic brain areas indicates a specific nutritional need for people and animals with epilepsy, as they are likely to benefit from auxiliary brain fuels other than glucose. Ketogenic diets provide the ketone bodies acetoacetate and ß-hydroxybutyrate, which can be used as auxiliary fuel by the brain. In approximately 50% children and adults with certain types of epilepsy, who can tolerate and maintain these dietary regimens, seizure frequency can be effectively reduced. More recent data demonstrate that addition of medium chain triglycerides (MCTs), which provide the medium chain fatty acids octanoic and decanoic acid, as well as ketone bodies as auxiliary brain energy, can be beneficial in rodent seizure models, and dogs and humans with epilepsy. Here, this evidence is reviewed, including tolerance in 65% of humans, efficacy studies in dogs, possible anticonvulsant mechanisms of actions of MCTs, and specifically decanoic acid as well as metabolic and antioxidant mechanisms. In conclusion, MCTs are a promising adjunct to standard pharmacological treatment for both humans and dogs with epilepsy, as they lack central nervous system side effects found with current antiepileptic drugs. There is now a need for larger clinical trials in children, adults, and dogs to find the ideal composition and doses of MCTs and the types of epilepsy that respond best.

Fructose 1,6-bisphosphate is anticonvulsant and improves oxidative glucose metabolism within the hippocampus and liver in the chronic pilocarpine mouse epilepsy model.

Glucose metabolism is altered in epilepsy, and this may contribute to seizure generation. Recent research has shown that metabolic therapies including the ketogenic diet and medium chain triglycerides can improve energy metabolism in the brain. Fructose 1,6-bisphosphate (F16BP) is an intermediate of glycolysis and when administered exogenously is anticonvulsant in several rodent seizure models and may alter glucose metabolism. Here, we showed that F16BP elevated the seizure threshold in the acute 6-Hz mouse seizure model and investigated if F16BP could restore impairments in glucose metabolism occurring in the chronic stage of the pilocarpine mouse model of epilepsy. Two weeks after the pilocarpine injections, mice that experienced status epilepticus (SE, "epileptic") and did not experience SE (no SE, "nonepileptic") were injected with vehicle (0.9% saline) or F16BP (1 g/kg in 0.9% saline) daily for 5 consecutive days. At 3 weeks, mice were injected with [U-13C6]-glucose and the % enrichment of 13C in key metabolites in addition to the total levels of each metabolite was measured in the hippocampal formation and liver. Fructose 1,6-bisphosphate increased total GABA in the hippocampal formation, regardless of whether mice had experienced SE. In the hippocampal formation, F16BP prevented reductions in the % 13C enrichment of citrate, succinate, malate, glutamate, GABA and aspartate that occurred in the chronic stage of the pilocarpine model. Interestingly, % 13C enrichment in glucose-derived metabolites was reduced in the liver in the chronic stage of the pilocarpine model. Fructose 1,6-bisphosphate was also beneficial in the liver, preventing reductions in % 13C enrichment of lactate and alanine that were associated with SE. This study confirmed that F16BP is anticonvulsant and can improve elements of glucose metabolism that are dysregulated in the chronic stage of the pilocarpine model, which may be due to reduction of spontaneous seizures. Our results highlight that F16BP may be therapeutically beneficial for epilepsies refractory to treatment.

Triheptanoin protects against status epilepticus-induced hippocampal mitochondrial dysfunctions, oxidative stress and neuronal degeneration.

Triheptanoin, the triglyceride of heptanoate, is anaplerotic (refills deficient tricarboxylic acid cycle intermediates) via the propionyl-CoA carboxylase pathway. It has been shown to be neuroprotective and anticonvulsant in several models of neurological disorders. Here, we investigated the effects of triheptanoin against changes of hippocampal mitochondrial functions, oxidative stress and cell death induced by pilocarpine-induced status epilepticus (SE) in mice. Ten days of triheptanoin pre-treatment did not protect against SE, but it preserved hippocampal mitochondrial functions including state 2, state 3 ADP, state 3 uncoupled respiration, respiration linked to ATP synthesis along with the activities of pyruvate dehydrogenase complex and oxoglutarate dehydrogenase complex 24 h post-SE. Triheptanoin prevented the SE-induced reductions of hippocampal mitochondrial superoxide dismutase activity and plasma antioxidant status as well as lipid peroxidation. It also reduced neuronal degeneration in hippocampal CA1 and CA3 regions 3 days after SE. In addition, heptanoate significantly reduced hydrogen peroxide-induced cell death in cultured neurons. In situ hybridization localized the enzymes of the propionyl-CoA carboxylase pathway, specifically Pccα, Pccß and methylmalonyl-CoA mutase to adult mouse hippocampal pyramidal neurons and dentate granule cells, indicating that anaplerosis may occur in neurons. In conclusion, triheptanoin appears to have anaplerotic and antioxidant effects which contribute to its neuroprotective properties.

The effects of C5aR1 on leukocyte infiltration following pilocarpine-induced status epilepticus.

This study aimed to determine the role C5aR1 plays in mediating immune responses acutely after pilocarpine-induced status epilepticus (SE), specifically those of brain-infiltrating leukocytes. Three days following pilocarpine SE, we determined by flow cytometry the brain immune cell phenotypes and measured key proinflammatory and antiinflammatory cytokine expression by infiltrating leukocytes and microglia in C5aR1-deficient and wild-type mice. Absence of C5aR1 reduced by 47% the numbers of Ly6G+ neutrophils in the brains of No-SE mice and decreased neutrophil entry after SE to levels found in wild-type brains that did not undergo SE (No-SE). Moreover, C5aR1-deficient mice showed increased interleukin (IL)-4 expression in infiltrating leukocytes, but not in microglia. Increases in IL-4 expression in infiltrating leukocytes coupled with decreased neutrophil invasion in C5aR1-deficient mice after SE is likely to contribute to the reduced neuronal loss previously found in these mice compared to their wild-type littermates. Although other SE models need to be investigated to substantiate our findings, this study provides further evidence that C5aR1 is an inflammatory mediator and may play a role in epileptogenesis.

Impaired hippocampal glucose metabolism during and after flurothyl-induced seizures in mice: Reduced phosphorylation coincides with reduced activity of pyruvate dehydrogenase.

OBJECTIVE: To determine changes in glucose metabolism and the enzymes involved in the hippocampus ictally and postictally in the acute mouse flurothyl seizure model. METHODS: [U-13 C]-Glucose was injected (i.p.) prior to, or following a 5 min flurothyl-induced seizure. Fifteen minutes later, mice were killed and the total metabolite levels and % 13 C enrichment were analyzed in the hippocampal formation using gas chromatography-mass spectrometry. Activities of key metabolic and antioxidant enzymes and the phosphorylation status of pyruvate dehydrogenase were measured, along with lipid peroxidation. RESULTS: During seizures, total lactate levels increased 1.7-fold; however, [M + 3] enrichment of both lactate and alanine were reduced by 30% and 43%, respectively, along with a 28% decrease in phosphofructokinase activity. Postictally the % 13 C enrichments of all measured tricarboxylic acid (TCA) cycle intermediates and the amino acids were reduced by 46-93%. At this time, pyruvate dehydrogenase (PDH) activity was 56% of that measured in controls, and there was a 1.9-fold increase in the phosphorylation of PDH at ser232. Phosphorylation of PDH is known to decrease its activity. SIGNIFICANCE: Here, we show that the increase of lactate levels during flurothyl seizures is from a source other than [U-13 C]-glucose, such as glycogen. Surprisingly, although we saw a reduction in phosphofructokinase activity during the seizure, metabolism of [U-13 C]-glucose into the TCA cycle seemed unaffected. Similar to our recent findings in the chronic phase of the pilocarpine model, postictally the metabolism of glucose by glycolysis and the TCA cycle was impaired along with reduced PDH activity. Although this decrease in activity may be a protective mechanism to reduce oxidative stress, which is observed in the flurothyl model, ATP is critical to the recovery of ion and neurotransmitter balance and return to normal brain function. Thus we identified promising novel strategies to enhance energy metabolism and recovery from seizures.

Alternative Fuels in Epilepsy and Amyotrophic Lateral Sclerosis.

This review summarises the recent findings on metabolic treatments for epilepsy and Amyotrophic Lateral Sclerosis (ALS) in honour of Professor Ursula Sonnewald. The metabolic impairments in rodent models of these disorders as well as affected patients are being discussed. In both epilepsy and ALS, there are defects in glucose uptake and reduced tricarboxylic acid (TCA) cycling, at least in part due to reduced amounts of C4 TCA cycle intermediates. In addition there are impairments in glycolysis in ALS. A reduction in glucose uptake can be addressed by providing the brain with alternative fuels, such as ketones or medium-chain triglycerides. As anaplerotic fuels, such as the triglyceride of heptanoate, triheptanoin, refill the TCA cycle C4/C5 intermediate pool that is deficient, they are ideal to boost TCA cycling and thus the oxidative metabolism of all fuels.

Modification of Astrocyte Metabolism as an Approach to the Treatment of Epilepsy: Triheptanoin and Acetyl-L-Carnitine.

Epilepsy is a severe neurological disorder characterized by altered electrical activity in the brain. Important pathophysiological mechanisms include disturbed metabolism and homeostasis of major excitatory and inhibitory neurotransmitters, glutamate and GABA. Current drug treatments are largely aimed at decreasing neuronal excitability and thereby preventing the occurrence of seizures. However, many patients are refractory to treatment and side effects are frequent. Temporal lobe epilepsy (TLE) is the most common type of drug-resistant epilepsy in adults. In rodents, the pilocarpine-status epilepticus model reflects the pathology and chronic spontaneous seizures of TLE and the pentylenetetrazole kindling model exhibits chronic induced limbic seizures. Accumulating evidence from studies on TLE points to alterations in astrocytes and neurons as key metabolic changes. The present review describes interventions which alleviate these disturbances in astrocyte-neuronal interactions by supporting mitochondrial metabolism. The compounds discussed are the endogenous transport molecule acetyl-L-carnitine and the triglyceride of heptanoate, triheptanoin. Both provide acetyl moieties for oxidation in the tricarboxylic acid cycle whereas heptanoate is also provides propionyl-CoA, which after carboxylation can produce succinyl-CoA, resulting in anaplerosis-the refilling of the tricarboxylic acid cycle.

Sulforaphane is anticonvulsant and improves mitochondrial function.

The nuclear factor erythroid 2-related factor 2 pathway (Nrf2) has been previously identified to protect the brain against various impacts. Here, we investigated the effect of the Nrf2 activator sulforaphane in various seizure models and hippocampal mitochondrial bioenergetics. We found that daily injections of sulforaphane for 5 days elevated the seizure thresholds to 6 Hz stimulation and fluorothyl-, but not pentylenetetrazole-induced tonic seizures and protected mice against pilocarpine-induced status epilepticus (SE). Also, sulforaphane increased the antioxidant defences within hippocampal formations and blood plasma. In addition, sulforaphane treatment reduced the extent of hippocampal lipid peroxidation 24 h post-SE and protected hippocampal mitochondria against SE-induced reduction in state 2 and uncoupler-stimulated state 3 respiration. SE-mediated partial loss of rotenone-sensitive and complex II-driven respiration was reduced, consistent with the enhanced activities of complexes I and II in sulforaphane-treated SE mice. In mitochondria isolated from both no SE and SE mice, sulforaphane increased state 3 respiration and respiration linked to ATP synthesis, which may contribute to its anticonvulsant and antioxidant effects by providing more ATP for cellular vital and protective functions. However, sulforaphane did not prevent SE-induced hippocampal cell death. In conclusion, sulforaphane and/or Nrf2 activation are viable anticonvulsant strategies, which are antioxidant and enhance mitochondrial function, especially the ability to produce ATP. Sulforaphane was anticonvulsant in two acute mouse models of epilepsy and protected mice against pilocarpine-induced status epilepticus (SE). We also found antioxidant effects of sulforaphane in mouse plasma and hippocampal formations, exhibited by increased catalase and superoxide dismutase (SOD) activity, as well as increased abilities of hippocampal mitochondria to produce ATP. These effects likely underlie sulforaphane's anticonvulsant mechanisms of action.

A novel anticonvulsant mechanism via inhibition of complement receptor C5ar1 in murine epilepsy models.

The role of complement system-mediated inflammation is of key interest in seizure and epilepsy pathophysiology, but its therapeutic potential has not yet been explored. We observed that the pro-inflammatory C5a receptor, C5ar1, is upregulated in two mouse models after status epilepticus; the pilocarpine model and the intrahippocampal kainate model. The C5ar1 antagonist, PMX53, was used to assess potential anticonvulsant actions of blocking this receptor pathway. PMX53 was found to be anticonvulsant in several acute models (6Hz and corneal kindling) and one chronic seizure model (intrahippocampal kainate model). The effects in the 6Hz model were not found in C5ar1-deficient mice, or with an inactive PMX53 analogue suggesting that the anticonvulsant effect of PMX53 is C5ar1-specific. In the pilocarpine model, inhibition or absence of C5ar1 during status epilepticus lessened seizure power and protected hippocampal neurons from degeneration as well as halved SE-associated mortality. C5ar1-deficiency during pilocarpine-induced status epilepticus also was accompanied by attenuation of TNFα upregulation by microglia, suggesting that C5ar1 activation results in TNFα release contributing to disease. Patch clamp studies showed that C5a-induced microglial K(+) outward currents were also inhibited with PMX53 providing a potential mechanism to explain acute anticonvulsant effects. In conclusion, our data indicate that C5ar1 activation plays a role in seizure initiation and severity, as well as neuronal degeneration following status epilepticus. The widespread anticonvulsant activity of PMX53 suggests that C5ar1 represents a novel target for improved anti-epileptic drug development which may be beneficial for pharmaco-resistant patients.

Complex alterations in microglial M1/M2 markers during the development of epilepsy in two mouse models.

OBJECTIVE: To characterize the changes in microglial proinflammatory M1 and antiinflammatory M2 marker expression during epileptogenesis in the chronic pilocarpine and intrahippocampal kainate models. METHODS: M1-activated microglia express proinflammatory cytokines driving infiltration of cells, whereas M2-activated microglia are more reparative, promoting phagocytosis of debris and expression of proteins associated with cellular stability and repair. Microglial markers were characterized as acute (3 days after status epilepticus [SE]), early chronic (21 days post-SE), and late chronic epileptic (5-12 months post-SE) time points. Following pilocarpine-SE, microglial markers were assessed by flow cytometry. Quantitative real-time polymerase chain reaction (RT-PCR) was used to measure messenger RNA (mRNA) levels of selected M1 (interleukin [IL] 1ß, tumor necrosis factor α [TNFα] cluster of differentiation [CD],CD16, and CD86), interleukin-6 [IL-6], interleukin-12 [IL-12], Fc receptors 16, and CD86) and M2 (arginase 1 [Arg1], chitinase-3-like protein [Ym1], found in inflammatory zone [FIZZ-1] [FIZZ-1], mannose receptor C type-1 [CD206], interleukin-4 [IL-4], and interleukin-10 (IL-10)) markers in both models. Video-electroencephalography (EEG) recordings were used to quantify late chronic seizure frequency. RESULTS: Three days post-SE microglia in the pilocarpine model expressed M1 and M2 markers, but only M1 markers were upregulated after kainate-induced SE. After 3 weeks, M1/M2 marker expression was largely ablated in the hippocampal formation of both models. Small mRNA level increases of CD11b, glial fibrillary acidic protein (GFAP), and IL-1ß were found in the pilocarpine model, consistent with IL-1ß contributing to spontaneous seizures, whereas mRNA levels of TNFα and Ym1 were decreased. In the late chronic phase, some M1/M2 markers, IL-1ß, TNFα, Arg1, Ym1, and CD206, resurged in the kainate, but not pilocarpine model, which may reflect and/or contribute to highly frequent seizures in kainate-SE mice. SIGNIFICANCE: The common M1 upregulation acutely post-SE may signal a role early in epileptogenesis, with a more pure "inflamed" central nervous system state after kainate-SE, potentially contributing to the development of more frequent seizures. The difference may also be due to the contribution of peripheral inflammation after pilocarpine injection. In summary, the microglial inflammatory response during epileptogenesis is complex, varies between models, and appears to correlate with chronic seizure frequency.

Triheptanoin partially restores levels of tricarboxylic acid cycle intermediates in the mouse pilocarpine model of epilepsy.

Triheptanoin, the triglyceride of heptanoate, is anticonvulsant in various epilepsy models. It is thought to improve energy metabolism in the epileptic brain by re-filling the tricarboxylic acid (TCA) cycle with C4-intermediates (anaplerosis). Here, we injected mice with [1,2-(13) C]glucose 3.5-4 weeks after pilocarpine-induced status epilepticus (SE) fed either a control or triheptanoin diet. Amounts of metabolites and incorporations of (13) C were determined in extracts of cerebral cortices and hippocampal formation and enzyme activity and mRNA expression were quantified. The percentage enrichment with two (13) C atoms in malate, citrate, succinate, and GABA was reduced in hippocampal formation of control-fed SE compared with control mice. Except for succinate, these reductions were not found in triheptanoin-fed SE mice, indicating that triheptanoin prevented a decrease of TCA cycle capacity. Compared to those on control diet, triheptanoin-fed SE mice showed few changes in most other metabolite levels and their (13) C labeling. Reduced pyruvate carboxylase mRNA and enzyme activity in forebrains and decreased [2,3-(13) C]aspartate amounts in cortex suggest a pyruvate carboxylation independent source of C-4 TCA cycle intermediates. Most likely anaplerosis was kept unchanged by carboxylation of propionyl-CoA derived from heptanoate. Further studies are proposed to fully understand triheptanoin's effects on neuroglial metabolism and interaction.

Vitamin B12 deficiency induces glucose intolerance, delays peak insulin levels and promotes ketogenesis in female rats.

Vitamin B12 (B12) deficiency is common among individuals with diabetes mellitus, but it is unknown if B12 deficiency contributes to impaired glucose homeostasis in this disorder. Female Sprague-Dawley rats were assigned to a control or B12-deficient diet for 4 weeks. Intraperitoneal glucose tolerance tests were performed after 25 days, and blood and liver samples were collected for metabolic profiling. B12 deficiency resulted in a prediabetic-like phenotype characterised by glucose intolerance, a delayed peak in plasma insulin levels following a glucose challenge and increased ketogenesis. We attributed increased ketogenesis to reduced liver anaplerosis, which limited the availability of the TCA cycle intermediates citrate, succinate and succinyl-CoA. This was associated with increased Mut mRNA levels and citrate synthase activity in the liver. One-carbon metabolite levels were altered in plasma and the liver, which was linked to reduced methylation capacity, altered amino acid levels and elevated Slc7a5 mRNA expression. Plasma folate and biotin levels were reduced, as were the majority of B vitamins in the liver. Changes in these B12-dependent processes and reduced B vitamin amounts likely contributed to deficits in glucose handling. Our findings highlight that B12 deficiency may promote the development of metabolic disorders like diabetes mellitus and emphasise the importance of adequate B12 intake for metabolic health.

Brain glycogen content is increased in the acute and interictal chronic stages of the mouse pilocarpine model of epilepsy.

Glucose is the main brain fuel in fed conditions, while astrocytic glycogen is used as supplemental fuel when the brain is stimulated. Brain glycogen levels are decreased shortly after induced seizures in rodents, but little is known about how glycogen levels are affected interictally in chronic models of epilepsy. Reduced glutamine synthetase activity has been suggested to lead to increased brain glycogen levels in humans with chronic epilepsy. Here, we used the mouse pilocarpine model of epilepsy to investigate whether brain glycogen levels are altered, both acutely and in the chronic stage of the model. One day after pilocarpine-induced convulsive status epilepticus (CSE), glycogen levels were higher in the hippocampal formation, cerebral cortex, and cerebellum. Opposite to expected, this was accompanied by elevated glutamine synthetase activity in the hippocampus but not the cortex. Increased interictal glycogen amounts were seen in the hippocampal formation and cerebral cortex in the chronic stage of the model (21 days post-CSE), suggesting long-lasting alterations in glycogen metabolism. Glycogen solubility in the cerebral cortex was unaltered in this epilepsy mouse model. Glycogen synthase kinase 3 beta (Gsk3b) mRNA levels were reduced in the hippocampal formations of mice in the chronic stage, which may underlie the elevated brain glycogen content in this model. This is the first report of elevated interictal glycogen levels in a chronic epilepsy model. Increased glycogen amounts in the brain may influence seizure susceptibility in this model, and this warrants further investigation.